Copper is an essential nutrient required in many cellular processes. It is a co-factor in anti-oxidant defense by superoxide dismutase, in neurotransmitter biosynthesis, in collagen production and in many other important biotransformations. Adequate copper is essential for human health, but elevated levels of copper are toxic. Sophisticated mechanisms have evolved to regulate the absorbtion and excretion of dietary copper and provide the important homeostasis of cellular and organismal copper. The major protein responsible for the high affinity uptake of copper into human cells is hCTRl The protein consists of 190 aminoacid residues and the PI of this application has, during the previous funding period characterized the membrane topology of hCTRl and shown that it is composed of three transmembrane segments separating an extracellular amino-terminus and an intracellular carboxy-terminus. Structure-function studies utilizing copper transport measurements and mutagenesis in insect cells suggested a pore-like mechanism for transport that has found support in the structural studies from the lab of another member of this program (Dr V Linger), these studies will be extended in mammalian cells. The present application extends a series of findings made in the Pi's lab during the previous funding period, and utilizes methods developed to study hCTRl in mammalian cells and epithelia. The PI has demonstrated that hCTRl is post-translationally modified with Olinked (as well as the more familiar N-linked) sugars. The O-linked modification protects hCTRl against a specific cellular proteolytic cleavage. The mechanisms involved in this specific cleavage and their role in the cell physiology of hCTRl will be characterized. In addition, it has been demonstrated that endogenous hCTRl is present at the basolateral surface of intestinal and renal epithelial cells and intestinal tissue. Thus an hCTR1-independent process is involved in the first step of copper acquisition from the diet. The proposed studies will identify and characterize this pathway as well as investigate the regulation of copper uptake in a number of cells. The roles of a second copper transporter, hCTR2, will also be explored. The experiments will provide essential information about how cells acquire their necessary copper.